table s1 analytical characteristics for all sterols shown ... lipolytica, erg accumulation under...
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Table S1 Analytical Characteristics for all sterols shown in the biosynthesis figure S1
Code IUPAC name Trivial name CAS number Chemical formula
MR
[g/mol] MR
(TMS) [g/mol]
RRT (cholestane)
Base peak (m/z)
Origin Reference
1*
(6E,10E,14E,18E)-2,6,10,15,19,23-Hexamethyl-tetracosa-2,6,10,14,18,22-
hexaene Squalene 7683-64-9 C30H50 410.7 (-) 0.95 69 R 2
2*
2,2-Dimethyl-3-((3E,7E,11E,15E)-3,7,12,16,20-pentamethylhenicosa-
3,7,11,15,19-pentaenyl)-oxirane Squalene epoxide 7200-26-2 C30H50O 426.7 (-) 1.05 81 R 2
3* 4,4,14-Trimethylcholesta-8,24(28)-dien-3β-
ol Lanosterol 79-63-0 C30H50O 426.7 498.9 1.44 393 R 1,2
4*
4,4-Dimethylcholesta-8,14,24-trien-3β-ol FF-MAS 64284-64-6 C29H44O 410.7 482.9 1.48 397 AF 1
5* 4,4-Dimethylcholesta-8,24-dien-3β-ol T-MAS 7448-02-4 C29H48O 412.7 484.9 1.47 379 AF 1
6 4-Methylcholesta-8,24-dien-3β-ol 4-Methylzymosterol C28H46O 398.7 470.9
7*
Cholesta-8,24-dien-3β-ol Zymosterol 128-33-6 C27H44O 384.7 456.9 1.31 351 R 1,2
8*
Ergosta-8,24(28)-dien-3β-ol Fecosterol 516-86-9 C28H46O 398.7 470.9 1.37 365 CG ERG2 (G)
9*
Ergosta-7,24(28)-dien-3β-ol Episterol 474-68-0 C28H46O 398.7 470.9 1.41 343 AF 1
10*
Ergosta-5,7,24(28)-trien-3β-ol 5-Dehydroepisterol 23582-83-4 C28H44O 396.7 468.9 1.39 363 SC ERG5 (I) 1
11*
Ergosta-5,7,22,24(28)-tetraen-3β-ol Dehydroergosterol 29560-24-5 C28H42O 394.7 466.8 1.36 361 CG ERG5 (I) 1
12*
Ergosta-5,7,22-trien-3β-ol Ergosterol 57-67-4 C28H44O 396.7 468.9 1.33 363 R 1
13*
4-Methylergosta-8,24(28)-dien-3β-ol 4-Methylfecosterol C29H48O 412.7 484.9 1.45 379 AF
14 Ergosta-8,22,24(28)-trien-3β‑ol C28H44O 396.7 468.9
15*
Ergosta-7,22-dien-3β‑ol 5-Dihydroergosterol/
Stellasterol 2465-11-4 C28H46O 398.7 470.9 1.35 343 R 1
16*
Ergosta-7,22,24(28)-trien-3β-ol C28H44O 396.7 468.9 1.37 343 AF 1
17*
Ergosta‑8,22‑dien‑3β‑ol 6673-68-3 C28H46O 398.7 470.9 1.32 365 SC ERG2 (G) 1
18*
Ergosta-8-en-3β-ol 30365-65-2 C28H48O 400.7 472.9 1.38 472 CG ERG2 (G) 1
19*
Ergosta-7-en-3β-ol 516-78-9 C28H48O 400.7 472.9 1.42 472 AF 1
20*
Ergosta-5,7-dien-3β-ol 516-79-0 C28H46O 398.7 470.9 1.39 365 YL ERG5 (I) 1
21*
Ergosta-5,8,24(28)-trien-3β‑ol C28H44O 396.7 468.9 1.36 363 AF
22*
Ergosta-5,8,22,24(28)-tetraen-3β-ol C28H42O 394.7 466.9 1.35 466 YL ERF2 (G)
23*
Ergosta-5,8,22-trien-3β-ol Lichesterol 50657-31-3 C28H44O 396.7 468.9 1.30 363 CG ERG2 (G) 1
24*
Ergosta-5,8-dien-3β‑ol C28H46O 398.7 470.9 1.36 339 R 3
25*
4,4,14-Trimethylergosta-8,24(28)-dien-3β-ol Eburicol 6890-88-6 C31H52O 440.7 512.9 1.50 407 CK ERG11 (C) 4
26*
4,14-Dimethylergosta-8,24(28)-dien-3β-ol Obtusifoliol C30H50O 426.7 498.9 1.43 393 AF ERG11 (C)
27*
14-Methylergosta-8,24(28)-dien-3β-ol 14-Methylfecosterol 33886-74-7 C29H48O 412.7 484.9 1.38 379 AF ERG11 (C) 1
28
4,4,14-Trimethylergosta-8-en-3β-ol C31H54O 442.7 514.9
Nature Protocols: doi:10.1038/nprot.2017.005
29 4,4,14-Trimethylergosta-8,22-dien-3β-ol C31H52O 440.7 512.9
30*
4,4,14-Trimethylergosta-7,22-dien-3β-ol C31H52O 440.7 512.9 1.48 407 CK ERG11 (C)
31 4,4,14-Trimethylergosta-8,22,24(28)-trien-
3β-ol C31H50O 438.7 510.9
32*
14-Methylergosta-8,24(28)-dien-3β,6α-diol 120523-06-0 C29H48O2 428.7 573.1 1.43 377 CK ERG11 (C) 1,4
33* 14-Methylergosta-8,22,24(28)-trien-3β,6α-
diol C29H46O2 426.7 571.1 1.39 123 CK ERG11 (C) 4
34*
14-Methylergosta-8,22-dien-3β,6α-diol C29H48O2 428.7 573.1 1.36 253 CK ERG11 (C) 4
35 14-Methylergosta-8-en-3β,6α-diol C29H50O2 430.7 575.1
36*
14-Methylergosta-7,24(28)-dien-3β,6α-diol C29H48O2 428.7 573.1 1.49 377 CK ERG11 (C) 4
37 14-Methylergosta-7,22,24(28)-trien-3β,6α-
diol C29H46O2 426.7 571.1
38 14-Methylergosta-7,22-dien-3β,6α-diol C29H48O2 428.7 573.1
39 14-Methylergosta-7-en-3β,6α-diol C29H50O2 430.7 575.1
40*
Cholesta-7,24-dien‑3β-ol 651-54-7 C27H44O 384.7 456.8 1.34 343 CG ERG6 (F) 1
41*
Cholesta-5,7,24‑trien‑3β-ol 7-Dehydrodesmosterol 1715-86-2 C27H42O 382.7 454.8 1.32 349 YL ERG6 (F) 1
42*
Cholesta-5,7,22,24‑tetraen‑3β-ol C27H40O 380.6 452.8 1.35 347 YL ERG6 (F) 2
43 Cholesta-8,22,24‑trien‑3β-ol C27H42O 382.6 454.8
44 Cholesta-7,22,24‑trien‑3β-ol C27H42O 382.6 454.8
45*
Cholesta-5-en-3ß-ol Cholesterol 57-88-5 C27H46O 386.6 458.6 1.25
(RT 14.35 min) 368 R 2
46*
Cholestane 481-20-9 C27H48 372.7 (-) 1.00
(RT 11.55 min) 217 R 2
AF Aspergillus fumigatus, CK Candida krusei, CG Candida glabrata, R synthetic or isolated reference material, RT retention time; RRT relative retention time, SC Saccharomyces cerevisiae, YL
Yarrowia lipolytica, ERG accumulation under specified enzyme inhibition (Enzyme code). * sterol can be found in spectral library. Sterol without * are only listed because of their appearance in the
biosynthesis figure 1, no analytical data is available.
1 Müller, C. et al. A convenient cellular assay for the identification of the molecular target of ergosterol biosynthesis inhibitors and quantification of their effects
on total ergosterol biosynthesis. Steroids 78, 483-493 (2013). 2 Giera, M., Müller, C. & Bracher, F. Analysis and experimental inhibition of distal cholesterol biosynthesis. Chromatographia 78, 343-58 (2014). 3 Giera, M. & Bracher, F. First total synthesis of ergosta-5,8-dien-3β-ol. Sci. Pharm. 76, 599-604 (2008). 4 Keller, P. et al. An antifungal benzimidazole derivative inhibits ergosterol biosynthesis and reveals novel sterols. Antimicrob. Agents Chemother. 59, 6296-6307
(2015).
Nature Protocols: doi:10.1038/nprot.2017.005
Table S2 Mass spectra of ergosterol biosynthesis intermediates. For all sterols, the free
alcohols are depicted, without stereo-chemical assignments
IUPAC name (6E,10E,14E,18E)-2,6,10,15,19,23-Hexamethyl-tetracosa-
2,6,10,14,18,22-hexaene
trival name Squalene
code 1 RRT 0.95
CAS number 7683-64-9 MR [g/mol] 410
chemical formula C30H50 MR (TMS)
[g/mol] (-)
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 2,2-Dimethyl-3-((3E,7E,11E,15E)-3,7,12,16,20-pentamethyl-
henicosa-3,7,11,15,19-pentaenyl)-oxirane
trival name Squalene epoxide
code 2 RRT 1.05
CAS number 7200-26-2 MR [g/mol] 426
chemical formula C30H50O MR (TMS)
[g/mol] (-)
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 4,4,14-Trimethylcholesta-8,24(28)-dien-3β-ol
trival name Lanosterol
code 3 RRT 1.44
CAS number 79-63-0 MR [g/mol] 426
chemical formula C30H50O MR (TMS)
[g/mol] 498
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 4,4-Dimethylcholesta-8,14,24(28)-trien-3β-ol
trival name FF-MAS
code 4 RRT 1.48
CAS number 64284-64-6 MR [g/mol] 410
chemical formula C29H44O MR (TMS)
[g/mol] 482
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 4,4-Dimethylcholesta-8,24-dien-3β-ol
trival name T-MAS
code 5 RRT 1.47
CAS number 7448-02-4 MR [g/mol] 412
chemical formula C29H48O MR (TMS)
[g/mol] 484
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Cholesta-8,24-dien-3β-ol
trival name Zymosterol
code 7 RRT 1.31
CAS number 128-33-6 MR [g/mol] 384
chemical formula C27H44O MR (TMS)
[g/mol] 456
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-8,24(28)-dien-3β-ol
trival name Fecosterol
code 8 RRT 1.37
CAS number 516-86-9 MR [g/mol] 398
chemical formula C28H46O MR (TMS)
[g/mol] 470
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-7,24(28)-dien-3β-ol
trival name Episterol
code 9 RRT 1.41
CAS number 474-68-0 MR [g/mol] 398
chemical formula C28H46O MR (TMS)
[g/mol] 470
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-5,7,24(28)-trien-3β-ol
trival name 5-Dehydroepisterol
code 10 RRT 1.39
CAS number 23582-83-4 MR [g/mol] 396
chemical formula C28H44O MR (TMS)
[g/mol] 468
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-5,7,22,24(28)-tetraen-3β-ol
trival name Dehydroergosterol
code 11 RRT 1.36
CAS number 29560-24-5 MR [g/mol] 394
chemical formula C28H42O MR (TMS)
[g/mol] 466
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-5,7,22-trien-3β-ol
trival name Ergosterol
code 12 RRT 1.33
CAS number 57-67-4 MR [g/mol] 396
chemical formula C28H44O MR (TMS)
[g/mol] 468
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 4-Methylergosta-8,24(28)-dien-3β-ol
trival name 4-Methylfecosterol
code 13 RRT 1.45
CAS number (-) MR [g/mol] 412
chemical formula C29H48O MR (TMS)
[g/mol] 484
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-8,22,24(28)-trien-3β-ol
trival name (-)
code 14 RRT 1.33
CAS number (-) MR [g/mol] 396
chemical formula C28H44O MR (TMS)
[g/mol] 468
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-7,22-dien-3β-ol
trival name 5-Dihydroergosterol/Stellasterol
code 15 RRT 1.35
CAS number 2465-11-4 MR [g/mol] 398
chemical formula C28H46O MR (TMS)
[g/mol] 470
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-7,22,24(28)-trien-3β-ol
trival name (-)
code 16 RRT 1.37
CAS number (-) MR [g/mol] 396
chemical formula C28H44O MR (TMS)
[g/mol] 468
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-8,22-dien-3β-ol
trival name (-)
code 17 RRT 1.32
CAS number (-) MR [g/mol] 398
chemical formula C28H46O MR (TMS)
[g/mol] 470
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-8-en-3β-ol
trival name (-)
code 18 RRT 1.38
CAS number 30365-65-2 MR [g/mol] 400
chemical formula C28H48O MR (TMS)
[g/mol] 472
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-7-en-3β-ol
trival name (-)
code 19 RRT 1.42
CAS number 516-78-9 MR [g/mol] 400
chemical formula C28H48O MR (TMS)
[g/mol] 472
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-5,7-dien-3β-ol
trival name (-)
code 20 RRT 1.39
CAS number 516-79-0 MR [g/mol] 398
chemical formula C28H46O MR (TMS)
[g/mol] 470
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-5,8,24(28)-trien-3β-ol
trival name (-)
code 21 RRT 1.36
CAS number (-) MR [g/mol] 396
chemical formula C28H44O MR (TMS)
[g/mol] 468
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC Name Ergosta-5,8,22,24(28)-tetraen-3β-ol
trival name (-)
code 22 RRT 1.35
CAS number (-) MR [g/mol] 394
chemical formula C28H42O MR (TMS)
[g/mol] 466
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-5,8,22-trien-3β-ol
trival name Lichesterol
code 23 RRT 1.30
CAS number 50657-31-3 MR [g/mol] 396
chemical formula C28H44O MR (TMS)
[g/mol] 468
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Ergosta-5,8-dien-3β-ol
trival name (-)
code 24 RRT 1.36
CAS number (-) MR [g/mol] 398
chemical formula C28H46O MR (TMS)
[g/mol] 470
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 4,4,14-Trimethylergosta-8,24(28)-dien-3β-ol
trival name Eburicol
code 25 RRT 1.50
CAS number 6890-88-6 MR [g/mol] 440
chemical formula C31H52O MR (TMS)
[g/mol] 512
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 4,14-Dimethylergosta-8,24(28)-dien-3β-ol
trival name Obtusifoliol
code 26 RRT 1.43
CAS number (-) MR [g/mol] 426
chemical formula C30H50O MR (TMS)
[g/mol] 498
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 14-Methylergosta-8,24(28)-dien-3β-ol
trival name 14-Methylfecosterol
code 27 RRT 1.38
CAS number 33886-74-7 MR [g/mol] 412
chemical formula C29H48O MR (TMS)
[g/mol] 484
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 4,4,14-Trimethylergosta-7,22-dien-3β-ol
trival name (-)
code 30 RRT 1.48
CAS number (-) MR [g/mol] 440
chemical formula C31H52O MR (TMS)
[g/mol] 512
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 14-Methylergosta-8,24(28)-dien-3β,6α-diol
trival name (-)
code 32 RRT 1.43
CAS number 120523-06-0 MR [g/mol] 428
chemical formula C29H48O2 MR (TMS)
[g/mol] 572
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 14-Methylergosta-8,22,24(28)-trien-3β,6α-diol
trival name (-)
code 33 RRT 1.39
CAS number (-) MR [g/mol] 426
chemical formula C29H46O2 MR (TMS)
[g/mol] 570
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 14-Methylergosta-8,22-dien-3β,6α-diol
trival name (-)
code 34 RRT 1.36
CAS number (-) MR [g/mol] 428
chemical formula C29H48O2 MR (TMS)
[g/mol] 572
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name 14-Methylergosta-7,24(28)-dien-3β,6α-diol
trival name (-)
code 36 RRT 1.49
CAS number (-) MR [g/mol] 428
chemical formula C29H48O2 MR (TMS)
[g/mol] 572
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Cholesta-7,24-dien-3β-ol
trival name (-)
code 40 RRT 1.34
CAS number 651-54-7 MR [g/mol] 384
chemical formula C27H44O MR (TMS)
[g/mol] 456
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Cholesta-5,7,24-trien-3β-ol
trival name 7-Dehydrodesmosterol
code 41 RRT 1.32
CAS number 1715-86-2 MR [g/mol] 382
chemical formula C27H42O MR (TMS)
[g/mol] 454
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Cholesta-5,7,22,24-tetraen-3β-ol
trival name (-)
code 42 RRT 1.35
CAS number (-) MR [g/mol] 380
chemical formula C27H40O MR (TMS)
[g/mol] 452
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Cholesta-5-en-3ß-ol
trival name Cholesterol
code 45 RRT 1.25
CAS number 57-88-5 MR [g/mol] 386
chemical formula C27H46O MR (TMS)
[g/mol] 458
Nature Protocols: doi:10.1038/nprot.2017.005
IUPAC name Cholestane
trival name Cholestane
code 46 RRT 1.00
CAS number 481-20-9 MR [g/mol] 372
chemical formula C27H48 MR (TMS)
[g/mol] (-)
Nature Protocols: doi:10.1038/nprot.2017.005
Table S3 – semi-quantitative results for the incubation of A. fumigatus and S. cerevisiae with different inhibitors
Sterol % of total area
3 9 10 12 20 22 23 25 sum
(I) 0.4 1.0 0.7 93.1 1.5 1.1 1.3 0.90 100
SD 0.06 0.0 0.06 0.15 0.0 0.06 0.1 0.0
(II) 2.5 0.7 0.3 73.4 2.2 1.2 1.0 18.6 100
SD 0.7 0.4 0.07 7.5 1.0 0.7 0.3 8.1
t-test * ns * * ns ns ns *
Fold Difference (II / I)
6.3 0.7 0.4 0.8 1.5 1.1 0.8 20.6
Table S3A Semi-quantitative evaluation of an enzyme inhibition in distal ergosterol biosynthesis (n=3 for control; n=5 for Posaconazole treatment) relative sterol composition of
total area(%), (I) Aspergillus fumigatus without treatment, (II) Aspergillus fumigatus under treatment with Posaconazole (0.5 µg/ml), marker sterols for an inhibition of the
sterol C14-demethylase in red (compare table 1 and figure 1&3), SD standard deviation, t-test – two-tailed Mann Whitney test, * p < 0.05; ** p < 0.01; ns not significant
Sterol % of total area 3 10 12 20 sum
(I) 9.3 0.0 78.7 12.0 100
SD 1.3 0.0 1.2 0.9
(II) 6.6 9.9 56.9 26.6 100
SD 1.7 0.4 5.7 4.0
t-test ns - p=0.07 p=0.08
Fold Difference (II / I) 0.7 - 0.7 2.2
Table S3B Semi-quantitative evaluation of an enzyme inhibition in distal ergosterol biosynthesis (n=3 for control; n=3 for 22-desaturase inhibitor treatment) relative sterol
composition of total area(%), (I) Saccharomyces cerevisiae without treatment, (II) Saccharomyces cerevisiae under treatment with a desaturase inhibitor (cmpd 4 in ref. 13) (0.5
µg/ml), marker sterols for an inhibition of the sterol C22-desaturase in red (compare table 1 and figure 1&3), SD standard deviation, t-test – two-tailed Mann Whitney test, * p
< 0.05; ** p < 0.01; ns not significant
Sterol % of total area 3 10 11 12 20 sum
(I) 9.3 0.0 0.0 78.7 12.0 100
SD 1.3 0.0 0.0 1.2 0.9
(II) 9.6 12.0 5.1 45.4 28.0 100
SD 0.6 1.7 0.4 5.4 3.6
t-test ns - - p=0.1 p=0.07
Fold Difference (II / I) 1.0 - - 0.6 2.3
Table S3C Semi-quantitative evaluation of an enzyme inhibition in distal ergosterol biosynthesis (n=3 for control; n=3 for 22-desaturase inhibitor treatment) relative sterol
composition of total area(%), (I) Saccharomyces cerevisiae without treatment, (II) Saccharomyces cerevisiae under treatment with a desaturase inhibitor (cmpd 4 in ref. 13)(1.0
µg/ml), marker sterols for an inhibition of the sterol C22-desaturase in red (compare table 1 and figure 1&3), SD standard deviation, t-test – two-tailed Mann Whitney test, * p
< 0.05; ** p < 0.01; ns not significant
Nature Protocols: doi:10.1038/nprot.2017.005